Members of the genus Chlamydia are bacterial obligate intracellular parasites of eukaryotic cells. They constitute an important group of pathogenic bacteria that are responsible for multiple medically significant conditions. The species Chlamydia trachomatis is comprised of at least fifteen serologically defined groups or serovars that are associated with human diseases. Trachoma, the world's leading cause of infectious blindness, is caused by serovars A, B, Ba, and C. Chlamydial sexually transmitted disease (STD) is the most common reportable disease in the United States. Serovars D though K are most commonly associated with STDs. The more serious sequelae of these diseases, blindness from trachoma and pelvic inflammatory disease from chlamydial STD, are immunopathological responses to chronic or repeated infections. While trachoma and sexually transmitted infections are primarily localized to the mucosal epithelium, a more systemic infection, lymphogranuloma venereum (LGV), caused by C. trachomatis serovars L1, L2, and L3, is also a sexually transmitted infection that causes inflammation of the inguinal lymph nodes. C. pneumoniae, is a common cause of community acquired pneumonia and is currently of interest due to possible associations with a variety of chronic diseases. C. psittaci is a zoonotic disease that infects many different types of poultry and livestock thus is of economic importance to agricultural industries and is occasionally transmitted to humans. Chlamydiae undergo their entire intracellular developmental cycle within a parasitophorous vacuole, termed an inclusion, that is unique among intracellular parasites. Chlamydiae are endocytosed into a tightly membrane-bound vesicle which grows throughout the developmental cycle to accommodate an increasing number of intracellular bacteria. The chlamydial inclusion, unlike vacuoles containing other intracellular pathogens, is not interactive with endocytic vesicular trafficking pathways but is instead fusogenic with an incompletely understood exocytic pathway which delivers sphingomyelin and cholesterol from the Golgi apparatus to the plasma membrane. Although all species of Chlamydia intersect this pathway, no other intracellular parasites have yet been found to similarly interact with this host vesicular trafficking pathway. Sequestration of chlamydiae within a vesicle that intersects an exocytic pathway is hypothesized to provide a unique, protected intracellular niche in which the chlamydiae replicate. Entry into this pathway is an active process on the part of the chlamydiae as both de novo transcription and translation are required. Virtually all of these interactions are specific and localized to the inclusion. This specificity strongly suggests modification of the exposed inclusion membrane. Examples of cis-acting modifications to the nascent inclusion membrane include: evasion of lysosomal fusion, interactions with microtubules to deliver the nascent inclusion to the peri-Golgi region and microtubule organizing center, initiation of fusion with exocytic vesicular traffic from the Golgi apparatus, and recruitment of, but not fusion with, recycling endosomes containing transferrin and its receptor. Many of these interactions are temporally associated with the exposure of inclusion membrane proteins to the host cell cytoplasm by a chlamydial type III secretion system. C. trachomatis expresses up to fifty predicted inclusion membrane proteins characterized by a long, bilobed hydrophobic domain of approximately 40 amino acids in length. Incs are exposed on the cytosolic face of the inclusion membrane and thus are likely candidates for factors controlling interactions with the host cell. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. Here we describe a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III dependent manner from Y. pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first thirty amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. We employed recently developed genetic tools to verify localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of 50 Incs identified 10 which were previously unverified Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by co-localization with p-Src. Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they are not Incs. Collectively our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will further our understanding of how this pathogen maintains its unique intracellular niche and mediates interactions with the host. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of non-fusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for chlamydia with a method for complementation of site-specific mutants.